PROJECT 2 ?? PROJECT SUMMARY/ABSTRACT Hepatitis B virus (HBV) chronically infects 250 - 350 million people worldwide, causing most liver cancers, the second leading cause of cancer death in men and a major cancer in women. HBV maintains these chronic infections and induces cancer by active replication through poorly understood pathways that traffic viral DNA and RNAs between the nucleus and cytoplasm, and direct successive interactions of key viral and cell factors in the cytoplasm for capsid assembly, reverse transcription, envelopment and secretion. In this project, we will build on our striking new HBV discoveries and newly developed advanced imaging and other research tools to advance key issues in HBV biology, thus providing essential foundations for critically needed new approaches to disrupt HBV chronic infections and thus block development of HBV-induced liver cancers. Aim 1 will define the sites and transport connections of key HBV capsid assembly, maturation and budding steps in the cytoplasm, including where HBV polymerase (Pol) and pregenomic RNA (pgRNA) interact and are encapsidated, and where capsids undergo successive steps in reverse transcription, interact with viral envelope proteins, and bud into the secretory pathway. The results will integrate powerfully with parallel cryo-EM studies that we are performing with our collaborator Adam Zlotnick (Indiana Univ.) of the nano-scale structures of maturing capsids at each matched step, revealing in a multi-scale vision of HBV replication how these structural changes are translated into cell biology effects. Aim 2 will yield the first full analysis of HBV cccDNA trafficking, maintenance, mitotic segregation and transcription in the nucleus, and the unprecedented pulsiform nuclear export of HBV pgRNA that we recently discovered. These results will provide foundations for rational development of treatments targeting cccDNA as the main genome reservoir of chronic infection, and pgRNA export as an increasingly attractive control point. Aim 3 will define the basis and functions of our exciting new discoveries that most HBV Pol localizes to mitochondria for roles independent of reverse transcription, and that Pol stimulates production of mitochondrially active ceramides that promote apoptosis and mitophagy. The results will inform if and how this association supports Pol?s multiple emerging functions in modulating mitochondrially-linked innate immune responses, which has potential implications for HBV control and for the chronic inflammation that contributes to HBV oncogenesis.